Selection of viral vectors by screening viral display peptide libraries is an auspicious approach to improve safety and efficiency of gene vectors. The screening of random AAV peptide libraries occurs via the amplification of viruses from a multitude of potential targeting peptides each presented within an AAV capsid that are internalized into target cells, mediated by the peptide displayed on their surface.The aim of this thesis was the selection of cell type- or tissue-directed gene vectors from random peptide libraries displayed on adeno-associated virus (AAV) and their characterization.Immature malignant blood progenitor cells causing acute myeloid leukemia (AML) are generally considered to be transduction-resistant to most conventional gene vectors. We screened random AAV serotype 2 peptide libraries on AML cells to select vector capsids with optimized leukemia transduction capacity. The screening revealed a distinct peptide sequence motif displayed on the selected viral capsids. The capsid mutant displaying the peptide NQVGSWS transduced the leukemia cell line Kasumi-1 with up to 90% efficiency, in contrast to vectors displaying a random unselected peptide (0.2% efficiency). Transduction assays on a panel of cell lines showed that the NQVGSWS capsid was able to overcome resistance to AAV-transduction especially in hematopoietic cancer cells. We further showed that NQVGSWS transduction of leukemia cells is independent of the primary attachment receptor heparin sulfate proteoglycan that is used for infection by wild-type AAV-2. Finally, leukemia targeted NQVGSWS-AAV vectors harboring a suicide gene conferred selective killing to Kasumi-1 AML cells. Therefore, we concluded that the selected vector capsids are a suitable and valuable tool to target therapeutic genes to AML cells.Screening AAV peptide libraries in vivo provides much more appropriate conditions to select for tissue-targeted gene vectors than mere cell-based in vitro approaches. In the second part of this thesis we developed a PCR-based amplification method allowing for adenovirus independent screening of AAV libraries. We performed in vivo selections applying several kinetic approaches in animals over multiple rounds after intravenous administration. The polyoma middle T-transgenic murine breast cancer and murine lung tissue were used as prototype targets. The peptide sequences of AAV clones yielded distinct sequence motifs unique for the target tissue. Selected capsid mutants conferred gene expression in the target tissue which was not detectable in animals injected with control vectors. However, most of the clones also transduced heart tissue in addition to the target tissue. We therefore conclude that this approach may be particularly useful if the tropism of the intended gene transfer in vivo has to be extended to rather than confined to the tissue of interest, indicating that targeting AAV to certain tissues in vivo seem to require more than one capsid modification. This impact the further development and improvement of AAV peptide libraries.Taken together, the work presented here demonstrates that random AAV displayed peptide libraries can be used to select for improved gene delivery vectors in vitro and, which is entirely novel, in vivo. Our results broaden the knowledge of transduction behavior of vectors isolated from AAV-2 libraries on different targets in vitro and in vivo and showed that such vectors have the potential to be used for therapeutic gene transfer.

engSelection and characterization of targeted vector capsids from random adeno-associated virus type 2 (AAV-2) display peptide librariesMichelfelder, Stefanterms-of-useSelektion und Charakterisierung zielgerichteter Vektorkapside aus randomisierten AAV-2 Peptidbanken2011-03-24T17:42:35Z2011-03-24T17:42:35ZMichelfelder, Stefanapplication/pdfSelection of viral vectors by screening viral display peptide libraries is an auspicious approach to improve safety and efficiency of gene vectors. The screening of random AAV peptide libraries occurs via the amplification of viruses from a multitude of potential targeting peptides each presented within an AAV capsid that are internalized into target cells, mediated by the peptide displayed on their surface.<br />The aim of this thesis was the selection of cell type- or tissue-directed gene vectors from random peptide libraries displayed on adeno-associated virus (AAV) and their characterization.<br />Immature malignant blood progenitor cells causing acute myeloid leukemia (AML) are generally considered to be transduction-resistant to most conventional gene vectors. We screened random AAV serotype 2 peptide libraries on AML cells to select vector capsids with optimized leukemia transduction capacity. The screening revealed a distinct peptide sequence motif displayed on the selected viral capsids. The capsid mutant displaying the peptide NQVGSWS transduced the leukemia cell line Kasumi-1 with up to 90% efficiency, in contrast to vectors displaying a random unselected peptide (0.2% efficiency). Transduction assays on a panel of cell lines showed that the NQVGSWS capsid was able to overcome resistance to AAV-transduction especially in hematopoietic cancer cells. We further showed that NQVGSWS transduction of leukemia cells is independent of the primary attachment receptor heparin sulfate proteoglycan that is used for infection by wild-type AAV-2. Finally, leukemia targeted NQVGSWS-AAV vectors harboring a suicide gene conferred selective killing to Kasumi-1 AML cells. Therefore, we concluded that the selected vector capsids are a suitable and valuable tool to target therapeutic genes to AML cells.<br />Screening AAV peptide libraries in vivo provides much more appropriate conditions to select for tissue-targeted gene vectors than mere cell-based in vitro approaches. In the second part of this thesis we developed a PCR-based amplification method allowing for adenovirus independent screening of AAV libraries. We performed in vivo selections applying several kinetic approaches in animals over multiple rounds after intravenous administration. The polyoma middle T-transgenic murine breast cancer and murine lung tissue were used as prototype targets. The peptide sequences of AAV clones yielded distinct sequence motifs unique for the target tissue. Selected capsid mutants conferred gene expression in the target tissue which was not detectable in animals injected with control vectors. However, most of the clones also transduced heart tissue in addition to the target tissue. We therefore conclude that this approach may be particularly useful if the tropism of the intended gene transfer in vivo has to be extended to rather than confined to the tissue of interest, indicating that targeting AAV to certain tissues in vivo seem to require more than one capsid modification. This impact the further development and improvement of AAV peptide libraries.<br />Taken together, the work presented here demonstrates that random AAV displayed peptide libraries can be used to select for improved gene delivery vectors in vitro and, which is entirely novel, in vivo. Our results broaden the knowledge of transduction behavior of vectors isolated from AAV-2 libraries on different targets in vitro and in vivo and showed that such vectors have the potential to be used for therapeutic gene transfer.2008